Novel peptide having angiotensin convertase inibitory effect

ABSTRACT

A peptide Met-Lys-Pro, which is obtained by chemical synthesis ot hydrolysis of casein, is used as the active ingredient of angiotensin converting enzyme inhibitors or hypotensive drugs.

TECHNICAL FIELD

The present invention relates to a novel peptide and an angiotensinconverting enzyme inhibitor containing the peptide. The angiotensinconverting enzyme inhibitor can be used as food products, feeding stuffsand pharmaceuticals.

BACKGROUND ART

Angiotensin converting enzyme (ACE) is an enzyme that acts onangiotensin I which is generated from angiotensinogen by digestion withrenin and converts it to angiotensin II by releasing two amino acids inthe C-terminal thereof. The angiotensin converting enzyme not only actson producing angiotensin II that has a strong hypertensive effect butalso acts on inactivating bradykinin that has a hypotensive effect. Fromthose actions, angiotensin converting enzyme inhibitors have been usedas therapeutic agents for hypertension, and, for example, Captopril(produced by Sankyo Co., Ltd.) and Renivase (produced by BanyuPharmaceutical Co., Ltd.) have been known as commercially availabledrugs. In addition, it is also known that the angiotensin convertingenzyme inhibitors have an effect of making cardiac hypertrophy regress.

On the other hand, peptides having angiotensin converting enzymeinhibitory effects have been found in natural products or inenzyme-degraded products of animal proteins such as casein and gelatin,vegetable proteins such as those from wheat, rice and corn, and fishproteins such as those from sardine. For instance, the known peptidesfound in natural products include teprotide (nonapeptide, SQ20881) andmetabolite IS83 of Actinomyces bacterium belonging to the genusStreptomyces (JP 58-177920 A). In addition, the known enzyme-degradedproducts include peptides obtained by decomposing casein with trypsin(JP 58-109425 A, JP 59-44323 A, JP 59-44324 A, JP 61-36226 A, and JP61-36227 A), peptides obtained by hydrolyzing casein with thermolysin(JP 6-277090 A, JP 6-277091 A, JP6-279491A, JP7-101982A, andJP7-101985A), peptides obtained by hydrolyzing casein or the like withlactic bacteria or a combination of proteinases and peptidases (JP6-197786 A, JP 6-40944 A, and JP 2001-136995 A (which are hereinafterreferred to as “References 1 to 3”, respectively) The peptides ofReferences 1 to 3 are used as foods for specified health use having thehypotensive effect.

Among the peptides described above, the peptide described in JP 7-101982A (hereinafter, referred to as “Reference 4”) shows the highestinhibitory activity against angiotensin converting enzyme and has acomparatively simple structure of tripeptide.

DISCLOSURE OF THE INVENTION

As described above, various angiotensin converting enzyme inhibitorypeptides have been known in the art. However, those peptides are stillinsufficient in inhibitory activity against angiotensin convertingenzyme as a function in food products. Thus, it has been desired toobtain a peptide derived from a natural product having a higherinhibitory activity against angiotensin converting enzyme and alsohaving a simple structure and to apply such a peptide to food products,pharmaceuticals, or the like.

The inventors of the present invention have made extensive studies tosolve the above problem, and as a result, they found out that hydrolysisof casein with a specific enzyme forms a novel peptide having a highinhibitory activity against angiotensin converting enzyme in thehydrolysate and the peptide has a sequence represented by Met-Lys-Pro,and thereby completed the present invention.

That is, the present invention relates to a peptide consisting ofMet-Lys-Pro (hereinafter also referred to as “peptide of the presentinvention”).

Further, the present invention provides an angiotensin converting enzymeinhibitor including a peptide consisting of Met-Lys-Pro as an effectiveingredient.

Further, the present invention provides a hypotensive agent including apeptide consisting of Met-Lys-Pro as an effective ingredient.

Hereinafter, the present invention will be described in detail.

The peptide of the present invention has a sequence represented byMet-Lys-Pro. In addition, the peptide of the present invention maybe thesalts of the peptide. In the present invention, Met denotes anL-methionine residue, Lys denotes an L-lysine residue and Pro denotes anL-proline residue.

The peptide of the present invention can be produced by hydrolyzing aprotein such as casein with an appropriate hydrolase.

Hereinafter, a method of hydrolyzing a protein with a hydrolase will beexemplified.

For the hydrolysis of a protein with an enzyme, although the manner oftreatment varies depending on properties of a protein, a materialprotein is dispersed in cold water or heated water and dissolved thereinwhen the protein is soluble. When the protein has poor solubility, it ismixed with hot water and homogenized while vigorously stirring.

The protein is not specifically limited as far as it contains a sequencerepresented by Met-Lys-Pro and produces the peptide of the presentinvention when it is digested with an appropriate hydrolase. Thus, anyprotein originated from an animal or bacteria may be used. Inparticular, a preferable protein is casein that is available in bulk.

It is desirable to sterilize a solution containing the protein at 70 to90° C. for approximately 15 seconds to 10 minutes in view of preventingthe deterioration by bacterial pollution.

Subsequently, it is preferable to adjust pH of the protein-containingsolution to optimum pH for a hydrolase used or proximal pH thereof byadding a basic agent or an acidic agent to the solution. The basic oracidic agent used in the method of the present invention may be anybasic or acidic agent as far as it is acceptable in food products orpharmaceuticals. Specific examples of the basic agents include sodiumhydroxide, potassium hydroxide, and potassium carbonate and the acidicagents include hydrochloric acid, citric acid, phosphoric acid, andacetic acid.

Next, a predetermined amount of a hydrolase is added to the proteinsolution to carry out a reaction at a temperature of approximately 10 to85° C. for 0.1 to 48 hours.

The hydrolase is preferably an endopeptidase, although not specificallylimited as far as the hydrolase can hydrolyze the protein to generatethe peptide of the present invention. The endopeptidases include aprotease originated from Bacillus bacteria and a protease originatedfrom animal pancreases. Those enzymes are commercially available.Preferable protease originated from Bacillus bacteria can be exemplifiedby Biopuraze sp-20 (manufactured by Nagase Biochemical Industry Inc.)and Protease N (manufactured by Amano Enzyme Inc.), while preferableprotease originated from animal pancreases can be exemplified by PTN6.0S(manufactured by Novozymes Japan Ltd.). The protease originated fromBacillus bacteria is desirably added at a rate of 100 to 5000 activeunits per 1 gram of protein. On the other hand, the protease originatedfrom animal pancreases is desirably added at a rate of 3000 to 8000active units per 1 gram of protein.

The hydrolase used in the present invention may be one kind of hydrolaseor a combination of two or more kinds. When two or more hydrolases areused, their enzyme reactions may be carried out simultaneously orindependently. In the present invention, particularly preferable is touse a mixture of Biopuraze sp-20, Protease N, and PTN6.0S.

A solution in which an enzyme is added is kept at an appropriatetemperature depending on the type of the enzyme, for example, 30 to 60°C., preferably 45 to 55° C. to initiate the hydrolysis of the protein.Regarding the reaction time of hydrolysis, the reaction is continueduntil a preferable decomposition rate is attained while thedecomposition rate of the reaction is monitored. For obtaining thepeptide of the present invention, the decomposition rate of 20 to 30% isdesirable.

As for a method of calculating the decomposition rate of the protein,the total nitrogen content of a sample is determined by the Kjeldahlmethod (The Japanese Society for Food Science and Technology, Ed., “FoodAnalysis Method”, page 102, KORIN Publishing Co., Ltd., 1984) and thecontent of formol nitrogen in the sample is determined by the formoltitration method (Manda et al., Ed., “Laboratory Manuals of FoodEngineering”, First Volume, page 547, Yokendo Co., Ltd., 1970), followedby calculating the decomposition rate with the following equation usingthose measurements.Decomposition rate (%)=(Formol nitrogen content/Total nitrogencontent)×100

The termination of the enzyme reaction is, for example, performed bydeactivation of the enzyme in the hydrolysis solution. It can be carriedout by heat deactivation using the general method. The conditions forsufficient deactivation can be suitably determined with respect to aheating temperature and a retention time of the heat deactivation inconsideration of a thermal stability of the enzyme used. For example, itcan be carried out in a temperature range of 80 to 130° C. for aretention time of 30 minutes to 2 seconds.

From the above hydrolysis solution, preferably, the peptide of thepresent invention is isolated and purified. The purification of thepeptide is generally performed by the same technique as one employed inthe purification of an oligopeptide, for example, by appropriatelycombining various kinds of chromatography including ion-exchangechromatography, absorption chromatography, reversed phasechromatography, distribution chromatography and gel filtrationchromatography, solvent precipitation, extracting by salting, anddistribution between two liquid phases etc. At the time of purifying thepeptide of the present invention, fractions containing objectivematerials can be determined based on an angiotensin converting enzymeinhibitory effect described below. Active ingredients in those fractionscan be identified by mass spectrometry.

Furthermore, the peptide of the present invention can be also producedby chemical synthesis. The chemical synthesis of the peptide of thepresent invention can be carried out by a liquid phase method or a solidphase method, which are generally used for the synthesis of anoligopeptide. The synthesized peptide is deprotected if required, andthen unreacted reagents, byproducts, and so on are removed. Such peptidesynthesis can be carried out using a commercially available peptidesynthesizer. The production of the target peptide can be confirmed basedon an angiotensin converting enzyme inhibitory effect.

The peptide of the present invention can be used as an effectiveingredient of an angiotensin converting enzyme inhibitor. The peptide ofthe present invention has an inhibitory effect on angiotensin convertingenzyme and a suppressing effect on bradykinin inactivation and exhibitsa hypotensive effect. Therefore, it can be used as a preventive agent ora therapeutic agent against various diseases derived from hypertension,such as cerebral hemorrhage, cerebral infarction, angina pectoris,myocardial infarction, and renal insufficiency, more specifically, itcan be used as a hypotensive agent or the like. In addition, it is knownthat the angiotensin converting enzyme inhibitor also has effects oninstinct hypertension whose cause is unknown, so that the peptide of thepresent invention is also expected to show a therapeutic or preventiveeffect on the instinct hypertension. In addition, it can be used as atherapeutic or preventive drug for other diseases such as cardiachypertrophy and angina illness, on which the angiotensin convertingenzyme inhibitor is considered to be effective.

The angiotensin converting enzyme inhibitor of the present invention maybe administered either orally or parenterally, but the oraladministration is preferable. The parenteral administration includesintravenous injection, intrarectal administration, and inhalation. Thepharmaceutical forms for the oral administration include a tablet form,a capsule form, a troche form, a syrup form, a granule form, a powderform, and an ointment form. Upon pharmaceutical formulation, in additionto a whey protein hydrolysate, other ingredients such as an excipient, apH regulator, a colorant, and a flavoring agent for drugs, which aregenerally used for the conventional pharmaceutical formulation, can beused. Furthermore, any drug known in the art or to be found out infuture, which has an angiotensin converting enzyme inhibitory effect canbe also used together.

The peptide of the present invention may be contained as an effectiveingredient in a food product and, as an embodiment of the angiotensinconverting enzyme inhibitor, processed into a food product having anangiotensin converting enzyme inhibitory effect. Irrespective of theforms of liquid, paste, solid, and powder etc., those food productsinclude: in addition to candies, fluid diets, and feeding stuffs(including those for pet animals), wheat flour products such as bread,macaroni, spaghetti, noodles, bread mix, french-fry mix, and breadcrumb; instant foods such as instant noodles, cup noodles, retort-packedfoods, prepared foods, canned foods, microwave meals, instant soup orstew, instant miso soup or Japanese clean soup, canned soup,freeze-dried foods and other instant foods; agricultural processedproducts such as canned farm products, canned fruits, jam or marmalade,pickles, boiled beans, agricultural dry foods, and cereals(grain-processed products); processed marine products such as cannedmarine products, fish hams or sausages, fish paste, marine productdainties, and tsukudanis; stock farm-processed products such as cannedstockbreeding products, pastes, and stockbreeding flesh hams orsausages; milk and milk products such as processed milk, milk beverages,yogurt, lactic acid bacteria beverages, cheese, ice cream, modified drymilk, cream and other milk products; fats and fatty oils such as butter,margarine, and vegetable oils; basic seasonings such as soy sauce, miso,sauces, tomato-processed seasonings, sweet cooking rice wines, andvinegar; complex seasonings and foods such as cooking mix, curry premix,bastes, dressings, noodle soups, spices and other complex seasonings;frozen foods such as material frozen foods, half-prepared frozen foods,and cooking-finished frozen foods; confectioneries such as caramels,candies, chewing gums, chocolates, cookies, biscuits, cakes, pies,snacks, crackers, Japanese sweets, rice biscuits, beans confectionery,desserts and other confectioneries; beverages of taste such ascarbonated drinks, natural fruit juices, fruit juice drink, soft drinkthat contains fruit juice, pulp drink, fruit drink that containsberries, vegetable drink, soybean milk, soybean milk drink, coffeedrink, tea drink, powder drink, concentrated drink, sports drink,nutrition drink, and alcoholic beverages and other beverages of taste;and other food products on the market such as baby foods, fish flour,and boiled rice with tea paste.

In the angiotensin converting enzyme inhibitor of the present invention,the content of the peptide of the present invention is preferably atleast 0.001% by weight with respect to the final composition of theangiotensin converting enzyme inhibitor.

The dosage of the angiotensin converting enzyme inhibitor of the presentinvention varies depending on ages, symptoms, and so on. In general,however, the inhibitor is administered at the dosage of 0.001 to 3000mg/day, preferably 0.01 to 30 mg/day, further, it may be administeredeither once a day or two or three times a day.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates results of an MS/MS analysis on a peptide of thepresent invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described in more detail withexamples.

EXAMPLE 1 Production of Peptide by Enzyme Degradation of Casein

<1> Enzyme Degradation of Casein

900 g of water was added to 100 g of commercially available casein(manufactured by New Zealand Dairy Board) and the casein was dispersedtherein well. Then, pH of the resultant solution was adjusted to 7.0 bythe addition of sodium hydroxide to dissolve the casein completely andthereby, an aqueous solution of casein at a concentration of about 10%was prepared. The aqueous solution of casein was heat-sterilized at 85°C. for 10 minutes and then adjusted to a temperature of 50° C., followedby adjusting the pH thereof to 9.5 by the addition of sodium hydroxide.After that, 100, 800 active units (1,200 active units per 1 gram ofprotein) of Biopuraze sp-20 (manufactured by Nagase Biochemical IndustryInc.), 168,000 active units (2,000 active units per 1 gram of protein)of Protease N (manufactured by Amano Enzyme Inc.), and 588,000 activeunits (7,000 active units per 1 gram of protein) of PTN6.0S(manufactured by Novozymes Japan Ltd.) were added to the solution toinitiate a hydrolysis reaction. When the decomposition rate of caseinreached to 24.1%, the enzyme was deactivated by heating at 80° C. for 6minutes to terminate the enzyme reaction, and followed by cooling to 10°C. This hydrolysis solution was extra-filtrated by an extra-filtrationmembrane (manufactured by Asahi Kasei Corporation) of a fractionmolecular weight of 3,000, and then it was concentrated and followed byfreeze-drying, which resulted in 85 g of a freeze-dried product.

<2> Isolation of Peptide by HPLC

The casein hydrolysate was separated by a reverse phase HPLC. Theconditions of the HPLC are described in HPLC Condition 1 describedbelow.

[HPLC Condition 1]

-   -   Column: CAPCELL PAK C18 (UG120, 5 μm) 20 mm I.D.×250 mm        (Shiseido Co., Ltd.)    -   Detection: UV 215 nm    -   Flow rate: 16 ml/min    -   Eluent A: 1% acetonitrile aqueous solution containing 0.05% TFA    -   Eluent B: 25% acetonitrile aqueous solution containing 0.05% TFA

Under the linear gradient condition from 100% of Eluent A to 100% ofEluent B after 40 minutes, a hydrolysate was separated. For each elutedfraction, the angiotensin converting enzyme inhibitory ability wasdetermined by the method described later. As a result, a peptide havingthe angiotensin converting enzyme inhibitory ability was eluted at aretention time of 22 minutes. For purifying the peptide, it was furtherpurified by HPLC. This condition is represented below as HPLC Condition2.

[HPLC Condition 2]

-   -   Column: CAPCELL PAK C18 (UG300, 5 μm) 2.0 mm I.D.×250 mm        (Shiseido Co., Ltd.)    -   Detection: UV 215 nm    -   Flow rate: 0.2 ml/min    -   Eluent A: 1% acetonitrile aqueous solution containing 0.05% TFA    -   Eluent B: 10% acetonitrile aqueous solution containing 0.05% TFA

Under the linear gradient condition from 100% of Eluent A to 100% ofEluent B after 15 minutes, the strong angiotensin converting enzymeinhibitory ability was observed at a peak of a retention time of 13minutes. The angiotensin I converting enzyme inhibitory ability at thispeak was such that IC50 [a concentration (μg/ml) required for inhibiting50% of the angiotensin converting enzyme activity]=0.18 μg/ml.

The compound in the above activity peak was identified by the proteinsequencer (Model-473A) of Applied Biosystems Ltd. As a result, it wasfound that the compound had a novel structure of Met-Lys-Pro.Furthermore, the molecular weight (M) was identified as 374.2 using themass spectrometer LCQ from Thermoquest Co., Ltd., and daughter ions ofm/z=260, 215, and 129 etc. were detected by the MS/MS analysis with aparent ion of m/z=375.2 (MH+) as shown in FIG. 1.

Consequently, it was elucidated that the structure of the peptide havingan angiotensin converting enzyme inhibitory ability wasH-Met-Lys-Pro-OH. 42.5 mg of a tripeptide Met-Lys-Pro was contained inthe freeze-dried product (85 g).

EXAMPLE 2 Chemical Synthesis of Peptide

Using the peptide synthesizer (Model 433A, Applied Biosystems Ltd.) andalso using Fmoc-L-Met (Applied Biosystems Ltd.), Fmoc-Lys (Boc) (AppliedBiosystems Ltd.), and Fmoc-Pro-TrtA-PEG Resin (Watanabe Kagaku KogyoK.K.) as raw materials, a tripeptide Met-Lys-Pro was synthesized by asolid phase synthesis method. The operation was performed according tothe manual from Applied Biosystems Ltd., followed by deprotection. Thepeptide was purified under HPLC Condition 1 described above. As a resultof the measurement of the angiotensin converting enzyme inhibitoryability using this material, almost the same value (IC50=0.19 μg/ml) asthat of one extracted from the casein-degraded product obtained inExample 1 was obtained.

The molecular weight (M) of the obtained tripeptide was measured as374.2 by the mass spectrometry. Almost the same spectrum as in FIG. 1was obtained by the MS/MS analysis with the parent ion of mz=375.2(MH+).

EXAMPLE 3 Angiotensin Converting Enzyme Inhibitory Effect of Peptide

The measurement of angiotensin converting enzyme inhibition wasperformed according to the Method of Cushman et al. [BiochemicalPharmacology vol. 20, pages 1637-1648 (1971)].

As samples, the peptides (Met-Lys-Pro) obtained in Example 1 and Example2, peptides (Val-Pro-Pro, Ile-Pro-Pro) described in References 1 to 3,and peptide (Leu-Leu-Trp) described in Reference 4 were used. Each ofthose peptides was chemically synthesized in the same way as in Example2.

The sample was dissolved in a 0.1M borate buffer (containing 0.3M NaCl,pH 8.3) and 0.08 ml thereof was then added in a tube. After that, 0.2 mlof an enzyme substrate (Hippuryl-histidyl-leucine, manufactured by SigmaCo., Ltd.) adjusted to 5 mM with the 0.1M borate buffer (containing 0.3MNaCl, pH 8.3) was added and then incubated at 37° C. for 3 minutes.Then, 0.02 ml of the rabbit lung angiotensin converting enzyme(manufactured by Sigma Co., Ltd.) adjusted to 0.1 U/ml by addingdistilled water was added and then reacted at 37° C. for 30 minutes.

Subsequently, the reaction was terminated by adding 0.25 ml of 1Nhydrochloric acid. Then, 1.7 ml of ethyl acetate was added and themixture was stirred vigorously for 20 seconds, and centrifugation wasperformed at 3000 rpm for 10 minutes, followed by collecting 1.4 ml ofan ethyl acetate layer. After removing a solvent by heating the obtainedethyl acetate layer, 1.0 ml of distilled water was added and theabsorption (228 nm absorbance) of the extracted hippuric acid wasmeasured and defined as an enzyme activity.

From the following equation, the inhibitory activity was calculated andthen the IC50 [the concentration (μg/ml or μM) required for inhibiting50% of angiotensin converting enzyme activity] was defined. The resultsare shown in Table 1.Inhibition rate=(A−B)/(A−C)×100%

A: Enzyme activity (228 nm absorbance) in the case of containing nosample (peptide).

B: Enzyme activity (228 nm absorbance) when the sample was added.

C: Enzyme activity (228 nm absorbance) when the enzyme and the samplewere not added. TABLE 1 Peptide IC 50 (μM) Peptide of Example 2(Met-Lys-Pro) 0.5 Val-Pro-Pro 6 Ile-Pro-Pro 4 Leu-Leu-Trp 2.2

EXAMPLE 4 Hypotensive Effect of Peptide on Animal

<1> Test Method

Twelve 10-week-old male SHR/Hos rats (purchased from Japan SLC, Inc.)were preliminary kept for 1 week and then the blood pressures of therats were measured using the non-invasive automatic sphygmomanometer forsmall animal (MK-2000, manufactured by Muromachi Kikai Co., Ltd.).

The rats were divided into two groups each including 6 animals based ona systolic blood pressure so that mean systolic blood pressure of eachgroup should be almost same value before administration. After that, therats were fasted for about 16 hours. For the test group, theenzyme-degraded product of casein obtained in the section <1> of Example1 was dissolved in water for injection and then orally administrated ata rate of 10 mL/kg body weight (100 mg/kg body weight for theenzyme-degraded product of casein and 0.05 mg/kg body weight for thepeptide (Met-Lys-Pro) of the present invention). The blood pressure ofthe rats was measured 2 hours after the administration. For the controlgroup, the same volume of water for injection was orally administeredinstead of the aqueous solution containing enzyme-degraded product ofcasein. The blood pressure of the rat was measured 2 hours after theadministration.

<2> Test Results

The results are shown in Table 2. As is evident from Table 2, a drop inblood pressure was observed in the test group. On the other hand, it wasnot observed in the control group. Therefore, it was found that theenzyme-degraded product of casein containing the peptide (Met-Lys-Pro)of the present invention had a hypotensive effect on an animal. TABLE 2Systolic blood pressure Systolic blood pressure before administration 2hours after administration (mmHg) (mmHg) Test group 182 140 Controlgroup 184 177

EXAMPLE 5 Hypotensive Effect of Peptide on Human

<1> Test Method

Nine subjects were male volunteers at the age of 30 or more and lessthan 58 who suffered from mild hypertension showing systolic bloodpressures of 140 to 165 mm Hg at the time of a screening test (medicalexamination by a doctor) 3 weeks before the initiation of uptake and whodid not receive any treatment with hypotensive agent.

The subjects were divided into the test sample-uptake group of fivesubjects and the control group of four subjects so that theblood-pressure values measured at the time of the screening test, ahabit of smoking and ages should be equalized between the groups.

3 g of the enzyme-degraded product of casein obtained in the section <1>of Example 1 (containing 1.5 mg of the peptide (Met-Lys-Pro) of thepresent invention) was taken as a test sample once a day, and 3 g ofdextrin was taken as a control once a day. Blood pressure measurement(0-3rd weeks) was performed at almost the same time.

The obtained numerical values (systolic blood pressure) are shown inTables 3 and 4.

The obtained numerical values (systolic blood pressure) were analyzedwith respect to the significant difference thereof by the one wayanalysis of variation (see, for example, Kiyoshi Ichihara, “Statisticsfor Bioscience”, Fifth Issue, Nankodo Co., Ltd., Nov. 20, 1991, p.150-151) with a significant level of 5% using the statistical analysissoftware SPSS (manufactured by SPSS Inc.). When there was a significantdifference in the values, mean values were compared by the Dunnett'smultiple comparison method (see, for example, Kei Takeuchi and other 13persons, Ed., “Statistics Dictionary”, Toyo Keizai Inc., Dec. 4, 1989,p. 399). TABLE 3 Results of test sample-uptake group (Systolic bloodpressure unit mmHg) Before administration No. (0 week) 1st week 2nd week3rd week 1 162 150 149 145 2 163 160 152 160 3 157 153 136 142 4 145 137147 137 5 145 133 139 127

TABLE 4 Results of control group (Systolic blood pressure unit mmHg)Before administration No. (0 week) 1st week 2nd week 3rd week 1 150 145139 144 2 153 145 136 156 3 149 137 134 135 4 146 143 148 137>Test Results

As the results of the analysis with the statistical analysis softwareSPSS, there was found no significant difference in the control group atall of the 1st, 2nd, and 3rd weeks after the administration with respectto before the administration (0 week), while the results were obtainedthat there were the significant differences at the 2nd and 3rd weeksafter the administration in the test sample-uptake group. Therefore, itwas proved that the enzyme-degraded product of casein containing thepeptide (Met-Lys-Pro) of the present invention had a hypotensive effecton human.

INDUSTRIAL APPLICABILITY

According to the present invention, there is provided a novel peptideuseful as an angiotensin converting enzyme inhibitor. The peptide of thepresent invention is derived from a natural product and shows lowtoxicity and high safety. Thus, the peptide of the present invention canalso be contained as the active ingredient in food products and, as anembodiment of the hypotensive agent, processed into food products havinghypotensive effects.

1. A peptide consisting of Met-Lys-Pro.
 2. An angiotensin convertingenzyme inhibitor comprising a peptide consisting of Met-Lys-Pro as aneffective ingredient.
 3. A hypotensive agent comprising a peptideconsisting of Met-Lys-Pro as an effective ingredient.